Abstract

A molecular understanding of drug resistance mechanisms enables surveillance of the effectiveness of new antimicrobial therapies during development and deployment in the field. We used conventional drug resistance selection as well as a regime of limiting dilution at early stages of drug treatment to probe two antimalarial imidazolopiperazines, KAF156 and GNF179. The latter approach permits the isolation of low-fitness mutants that might otherwise be out-competed during selection. Whole-genome sequencing of 24 independently derived resistant Plasmodium falciparum clones revealed four parasites with mutations in the known cyclic amine resistance locus (pfcarl) and a further 20 with mutations in two previously unreported P. falciparum drug resistance genes, an acetyl-CoA transporter (pfact) and a UDP-galactose transporter (pfugt). Mutations were validated both in vitro by CRISPR editing in P. falciparum and in vivo by evolution of resistant Plasmodium berghei mutants. Both PfACT and PfUGT were localized to the endoplasmic reticulum by fluorescence microscopy. As mutations in pfact and pfugt conveyed resistance against additional unrelated chemical scaffolds, these genes are probably involved in broad mechanisms of antimalarial drug resistance.

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Acknowledgements

M.Y.L. is supported by the Economic Development Board—Industrial Postgraduate Programme (EDB-IPP) scholarship. G.L. is supported by an A.P. Giannini Post-Doctoral Fellowship. Work at UCSD was supported by grants from the National Institutes of Health (NIH; R01 AI090141 and R01 AI103058) to E.A.W. D.A.F. acknowledges support from the Medicines for Malaria Venture. B.M. and L.R. are supported by the Singapore Immunology Network under the Agency for Science, Technology and Research (A*STAR, Singapore). R.W. is a Research Associate at the National Fund for Scientific Research FNRS-FRS (Belgium). The authors thank C. Jensen (Leiden University Medical Center, Netherlands) for the donation of the P. berghei ANKA strain.

Author information

Author notes

    • Michelle Yi-Xiu Lim
    •  & Gregory LaMonte

    These authors contributed equally to this work.

Affiliations

  1. Novartis Institute for Tropical Diseases, 138670 Singapore

    • Michelle Yi-Xiu Lim
    • , Bee Huat Tan
    • , Bianca F. Tjahjadi
    • , Adeline Chua
    • , Peter Gedeck
    • , Ghislain M. C. Bonamy
    • , Bryan K. S. Yeung
    • , Liting Lim
    • , Thierry T. Diagana
    •  & Pablo Bifani
  2. Department of Pharmacy, Faculty of Science, National University of Singapore, 119077 Singapore

    • Michelle Yi-Xiu Lim
    •  & Paul Chi-Lui Ho
  3. Department of Pediatrics, School of Medicine, University of California, San Diego, La Jolla, California 92093, USA

    • Gregory LaMonte
    • , Christin Reimer
    • , Victoria Corey
    • , Marie Nachon
    •  & Elizabeth A. Winzeler
  4. Department of Microbiology & Immunology, Columbia University Medical Center, New York, New York 10032, USA

    • Marcus C. S. Lee
    •  & David A. Fidock
  5. Malaria Programme, Wellcome Trust Sanger Institute, Wellcome Genome Campus, Cambridge CB10 1SA, UK

    • Marcus C. S. Lee
  6. Department of Microbiology & Immunology, Yong Loo Lin School of Medicine, National University of Singapore, National University Health System, 117545 Singapore

    • Bianca F. Tjahjadi
    • , Benoit Malleret
    • , Thierry T. Diagana
    •  & Pablo Bifani
  7. Laboratory of Biopolymers and Supramolecular Nanomaterials, Faculty of Pharmacy, Université Libre de Bruxelles, Brussels, Belgium

    • René Wintjens
  8. Singapore Immunology Network (SIgN), A*Star, 138648 Singapore

    • Benoit Malleret
    •  & Laurent Renia
  9. Center for Advanced Technology, Department of Biochemistry and Biophysics, University of California, San Francisco, California 94143, USA

    • Eric D. Chow
  10. Division of Infectious Diseases, Department of Medicine, Columbia University Medical Center, New York, New York 10032, USA

    • David A. Fidock

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Contributions

M.Y.-X.L., G.L., M.C.S.L., E.A.W. and P.B. designed the experiments. M.Y.-X.L., G.L., M.C.S.L., C.R., B.H.T., V.C., B.F.T., A.C., M.N., B.M., E.D.C. and L.L. performed the experiments. Modelling work was performed by R.W. M.Y.-X.L., G.L., M.C.S.L., C.R., V.C., M.N., E.D.C. and P.G. analysed the data. G.M.C.B., P.C.-L.H., L.R., D.A.F. and T.T.D. contributed support. M.Y.-X.L., G.L., M.C.S.L., B.K.S.Y., D.A.F., E.A.W. and P.B. wrote and proofread the manuscript. E.A.W. and P.B. gave technical support and conceptual advice. The manuscript was edited by all authors.

Competing interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to Elizabeth A. Winzeler or Pablo Bifani.

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DOI

https://doi.org/10.1038/nmicrobiol.2016.166

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